Fungal - CBS-KNAW Fungal Biodiversity Centre

Fungal Diversity
Pseudocercospora flavomarginata sp. nov., from Eucalyptus
leaves in Thailand
Gavin C. Hunter1*, Pedro W. Crous1,2, Brenda D. Wingfield3, Krishna
Pongpanich4 and Michael J. Wingfield1
1
Department of Microbiology, Forestry and Agricultural Biotechnology Institute (FABI),
Faculty of Natural and Agricultural Science, University of Pretoria, Pretoria 0002, South
Africa
2
Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, P.O. Box 85167, 3508
AD, Utrecht, The Netherlands
3
Department of Genetics, University of Pretoria, Pretoria, 0002, South Africa
4
Forest Entomology and Microbiology Group, National Park, Wildlife and Plant Conservation
Department, Chatuchak, Bangkok, 10900, Thailand
Hunter, G.C., Crous, P.W., Wingfield, B.D., Pongpanich, K. and Wingfield, M.J. (2006).
Pseudocercospora flavomarginata sp. nov. from Eucalyptus leaves in Thailand. Fungal
Diversity 22: 71-90.
Mycosphaerella represents one of the largest ascomycete genera accommodating more than
3000 names. Approximately 60 Mycosphaerella species have been linked to leaf diseases on
Eucalyptus species, collectively known as Mycosphaerella Leaf Disease (MLD). Many
hyphomycete and coelomycete anamorph genera are linked to Mycosphaerella and several
species of the hyphomycete genus Pseudocercospora are associated with MLD symptoms on
various Eucalyptus species. Eucalyptus trees in Vietnam and Thailand, particularly those of E.
camaldulensis and hybrids of this species, commonly have a leaf spot disease caused by a
species of Pseudocercospora. Lesions associated with this disease are very characteristic, with
chlorotic margins and masses of brown conidiophores occurring predominantly on the abaxial
lesion surface. The aim of this study was to characterise the Pseudocercospora species
associated with this disease. This was achieved through studying the morphology of the fungus
and via DNA sequence analysis from four nuclear gene regions. Results showed that the
fungus represents an undescribed species of Pseudocercospora, that is formally described here
as Pseudocercospora flavomarginata.
Key words: Ascomycete, Eucalyptus leaf disease, Pseudocercospora
Introduction
Species of Eucalyptus are currently some of the most popular tree species
for commercial forestry, with plantations expanding more rapidly than for any
other tree species (Turnbull, 2000). This is largely due to their favourable
*
Corresponding author: Gavin C. Hunter; e-mail: [email protected]
71
wood qualities, relatively rapid growth and their adaptability to a very wide
range of different environments (Turnbull, 2000). Eucalyptus spp. are thus
propagated in commercially productive plantations in many tropical and subtropical countries, where timber is used primarily for the paper and pulp
industry. In South-East Asia, the forestry industry is expanding rapidly, and it
has been estimated that there are approximately 2 million ha of Eucalyptus
plantations in the area (Old et al., 2003).
The genus Mycosphaerella is one of the largest genera of Ascomycetes,
accommodating more than 3000 names (Corlett, 1991, 1995; Crous, 1998;
Aptroot, 2006). Approximately 60 Mycosphaerella species have been
associated with diseases on Eucalyptus leaves, collectively known as
Mycosphaerella Leaf Disease (MLD) (Crous, 1998; Maxwell et al., 2003;
Crous et al., 2004, 2006; Hunter et al., 2004, 2006). Symptoms of MLD
include leaf spots, defoliation, twig cankers and in severe cases stunting of tree
growth (Park and Keane, 1982; Carnegie and Keane, 1994; Crous, 1998).
The teleomorph state, represented by abundant small pseudothecia, is
most commonly associated with MLD on Eucalyptus leaves (Crous, 1998).
Mycosphaerella is, however, linked to approximately 23 anamorph genera
including both coelomycetes and hyphomycetes (Crous et al., 2000; Crous and
Braun, 2003; Kirschner et al., 2004; Schubert and Braun, 2005). Recent
surveys of Eucalyptus foliage in many parts of the world have led to a
significant increase in the number of Mycosphaerella spp. found on these trees
(Crous et al., 2004, 2006).
The anamorphic genus Pseudocercospora is large and morphologically
diverse (Crous et al., 2000). Species of Pseudocercospora occur on many plant
hosts where they cause leaf spots (Crous and Braun, 1996; Crous et al., 1997;
Crous and Braun, 2001; Braun and Dick, 2002). These fungi typically have
dematiaceous conidiophores and scolecosporous conidia with inconspicuous
conidial scars and conidiogenous cells that proliferate sympodially and
percurrently (Crous and Wingfield, 1997; Crous et al., 2000; Crous and Braun,
2003).
A very distinct leaf spot disease especially on Eucalyptus camaldulensis
and hybrids of this and other species is well known in Thailand and Vietnam
(Old et al., 2003). The symptoms of this disease are very obvious and not
easily confused with other leaf spots. Based on the occurrence of abundant
conidiophores that are found on the lesion, it is well recognised that the disease
is caused by a species of Pseudocercospora (Old et al., 2003). The fungus has,
however, never been critically compared with other Mycosphaerella spp.
occurring on Eucalyptus, nor has it been formally named. The aim of this study
was, therefore, to characterise the fungus and to provide a name for it. This was
72
Fungal Diversity
achieved through critical study of its morphological characteristics by
comparison to other Pseudocercospora spp. known to occur on Eucalyptus
(Crous, 1998; Braun and Dick, 2002) and via comparisons of DNA sequences
for the Large Subunit (LSU) and Internal Transcribed Spacer (ITS) region of
the rRNA operon, the Actin (ACT) and Translation Elongation Factor 1-alpha
(EF-1α) gene regions.
Materials and methods
Sample collection and fungal isolations
Leaf spots on Eucalyptus camaldulensis and hybrids of this species were
collected from trees growing in plantations in various parts of Thailand.
Diseased leaves showing the typical lesions with very distinct chlorotic
margins and bearing conidiophores of a Pseudocercospora sp. were collected
for subsequent laboratory study.
Leaf lesions were examined under a dissection microscope for the
presence of Pseudocercospora conidiophores. A sterile inoculation needle was
used to scrape conidia from the lesions and these were spread onto 2% MEA
agar plates (wt/v) (malt extract agar) (Biolab, Johannesburg, South Africa) and
incubated at 25°C. Subsequently, single germinating conidia were lifted from
the plates and transferred to fresh 2% MEA agar plates. Agar plates were
incubated at 25°C in the dark for 21 days to allow for culture growth. Isolates
of Mycosphaerella species occurring on Eucalyptus and known to have
Pseudocercospora anamorphs were also included in this study for comparative
purposes (Table 1). These isolates were all grown on 2% MEA agar plates for
approximately 1 month to ensure sufficient mycelial growth. All cultures used
during this study are maintained in the culture collection (CMW) of the
Forestry and Agricultural Biotechnology Institute (FABI) of the University of
Pretoria, Pretoria, South Africa. Representative cultures have also been
deposited with the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The
Netherlands (Table 1).
DNA extraction
Mycelia were scraped from actively growing cultures, freeze dried for 24
hours, and then ground into a fine powder using liquid nitrogen. DNA was
isolated according to the method of Hunter et al. (2004). A 1 : 1 Phenol :
Chloroform extraction was used. DNA was precipitated by the addition of
absolute ethanol (100% EtOH). Isolated DNA was cleaned by the addition of
73
Table 1. Isolates of Mycosphaerella and Pseudocercospora used for DNA sequencing and phylogenetic analysis.
Isolate No.
CMW CPC
4944 1106
11255 1104
3042 800
3033 802
9095 N/A
9096 N/A
14455 730
4942 760
14776 N/A
3046 1312
4943 1360
5223 1362
14458 3745
14778 822
14786 823
14914 1202
14785 1203
5148 N/A
5228 16
14784 1264
14779 1459
14908 10390
14911 10500
5151 1269
13586 N/A
13594 N/A
13590 N/A
14906 825
CBS
110969
110967
N/A
681.95
N/A
N/A
243.94
110682
111364
111190
114774
N/A
112895
110750
110693
114664
111280
N/A
110777
111070
111286
114242
114243
111175
118841
118824
118823
110748
Teleomorph
(Mycosphaerella)
M. colombiensis
M. colombiensis
M. crystallina
M. crystallina
M. fori
M. fori
M. gracilis
M. heimii
M. heimioides
M. heimioides
M. irregulariramosa
M. irregulariramosa
M. madeirae
M. marksii
M. marksii
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
M. lateralis
Anamorph
Origin
GenBank Accession No.
LSU
ITS
P. colombiensis
E. urophylla, Colombia
DQ204744 AY752149
P. colombiensis
E. urophylla, Columbia
DQ204745 AY752147
P. crystallina
E. bicostata, South Africa
DQ204746 DQ267578
P. crystallina
E. bicostata, South Africa
DQ204747 AY490757
Pseudocercospora sp. E. grandis, South Africa
DQ204748 AF468869
Pseudocercospora sp. E. grandis, South Africa
DQ204749 DQ267581
P. gracilis
E. urophylla, Indonesia
DQ204750 DQ267582
P. heimii
Eucalyptus sp., Madagascar
DQ204751 AF309606
P. heimioides
Eucalyptus sp., Indonesia
DQ204752 DQ267586
P. heimioides
Eucalyptus sp., Indonesia
DQ204753 AF309609
P. irregulariramosa
E. saligna, South Africa
DQ204754 AF309607
P. irregulariramosa
E. saligna, South Africa
DQ204755 AF309608
Pseudocercospora sp. E. globulus, Madeira
DQ204756 AY725553
P. epispermogonia
E grandis×saligna, South Africa DQ204757 DQ267596
P. epispermogonia
E grandis×saligna, South Africa DQ204758 DQ267597
P. basitruncata
E. grandis, Colombia
DQ204759 DQ267600
P. basitruncata
E. grandis, Colombia
DQ204760 DQ267601
P. basiramifera
E. pellita, Thailand
DQ204761 AF309595
P. eucalyptorum
E. nitens, South Africa
DQ204762 AF309598
P. natalensis
E. nitens, South Africa
DQ204763 AF309594
P. paraguayensis
E. nitens, Brazil
DQ204764 DQ267602
P. pseudoeucalyptorum E. globulus, Spain
DQ204765 AY725526
P. pseudoeucalyptorum E. nitens, New Zealand
DQ204766 AY725527
P. robusta
E. robusta, Malaysia
DQ204767 AF309597
P. flavomarginata
E. camaldulensis, Thailand
DQ153306 DQ155657
P. flavomarginata
E. camaldulensis, Thailand
DQ153308 DQ155658
P. flavomarginata
E. camaldulensis, Thailand
DQ153307 DQ155659
Dissoconium dekkeri
E. grandis×saligna, South Africa DQ204768 AF173315
ACT
DQ147639
DQ147640
DQ147637
DQ147636
DQ147618
DQ147619
DQ147616
DQ147638
DQ147632
DQ147633
DQ147634
DQ147635
DQ147641
DQ147629
DQ147628
DQ147622
DQ147621
DQ147607
DQ147614
DQ147623
DQ147606
DQ147613
DQ147615
DQ147617
DQ166513
DQ166514
DQ166515
DQ147651
EF-1α
DQ211660
DQ211661
DQ211662
DQ211663
DQ211664
DQ211665
DQ211666
DQ211667
DQ211668
DQ211669
DQ211670
DQ211671
DQ211672
DQ211673
DQ211674
DQ211675
DQ211676
DQ211677
DQ211678
DQ211679
DQ211680
DQ211681
DQ211682
DQ211683
DQ156548
DQ156549
DQ156550
DQ211684
CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
CMW: Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.
CPC: Working collection of Pedro Crous, formerly in STE-U, now housed at CBS. Isolate numbers from Crous (1998).
N/A: Not available.
Fungal Diversity
70% ethanol (70% EtOH) and dried under vacuum. Sterile water was used to
resuspend the isolated DNA. RNaseA (10 µg/µL) was added to the
resuspended DNA and incubated at 37°C for approximately 2 hours to digest
any residual RNA. Isolated DNA was visualised in a 1% agarose gel (wt/v)
(Roche Diagnostics, Mannheim, Germany) stained with ethidium bromide.
PCR amplification and purification
DNA (ca. 20 ng) isolated from the unknown Pseudocercospora sp. and
representative Mycosphaerella isolates that were used for comparative
purposes, was used as a template for amplification with the polymerase chain
reaction (PCR). For the purposes of this study, four nuclear gene regions were
chosen for amplification and subsequent DNA sequencing. These included the
internal transcribed spacer (ITS) region and the large subunit (LSU) of the
rRNA operon, a portion of the translation elongation factor 1-alpha gene (EF1α) and a portion of the actin (ACT) gene regions.
All PCR reaction mixtures for the four gene regions were performed in a
total volume of 25 µL containing 10 × PCR Buffer (5 mM Tris-HCl, 0.75 mM
MgCl2, 25 mM KCl, pH 8.3) (Roche Diagnostics, Johannesburg, South
Africa), 2.5 mM of each dNTP (dATP, dTTP, dCTP, dGTP) (Roche
Diagnostics, Johannesburg, South Africa), 0.2 µM of forward and reverse
primers (Inqaba Biotech, Pretoria, South Africa), 1.25 U Taq DNA Polymerase
(Roche Diagnostics, Johannesburg, South Africa) and DNA (20 ng/µL).
Sterilised distilled water was added to achieve a final volume of 25 µL.
The ITS 1, ITS 2 and the 5.8 S gene regions of the internal transcribed
spacer (ITS) region of the rRNA operon were amplified using primers ITS-1
(5`−TCC GTA GGT GAA CCT GCG G -3`) and LR-1 (5`-GGT TGG TTT
CTT TTC CT-3`) (Vilgalys and Hester, 1990; White et al., 1990). PCR
reaction conditions for the ITS followed those of Crous et al. (2004) and
Hunter et al. (2004). A portion of the large subunit (containing domain
D1−D3) of the nuclear rRNA operon was amplified using primers LROR (5’ACC CGC TGA ACT TAA GC-3’) (Moncalvo, Wang and Hseu, 1995) and
LR7 (5’-TAC TAC CAC CAA GAT CT-3’) (Vilgalys and Hester, 1990). PCR
reaction conditions were as follows: an initial denaturation temperature of
96°C for 2 minutes, followed by 35 cycles of denaturation at 94°C for 30
seconds, primer annealing at 62°C for 30 seconds, primer extension at 72°C for
1 minute and a final elongation step at 72°C for 7 minutes.
A portion of the translation elongation factor 1-alpha gene (EF-1α) was
amplified using the primers EF1-728F (5’-CAT CGA GAA GTT CGA GAA
GG-3’) and EF1-986R (5’-TAC TTG AAG GAA CCC TTA CC-3’) (Carbone
75
and Kohn, 1999). Reaction conditions were as follows: an initial denaturation
temperature of 96°C for 2 minutes, followed by 35 cycles of denaturation at
94°C for 30 seconds, primer annealing at 56°C for 30 seconds and primer
extension at 72°C for 30 seconds. The reaction was completed with a final
extension at 72°C for 7 minutes.
A portion of the actin (ACT) gene was amplified using the primers ACT512F (5’-ATG TGC AAG GCC GGT TTC GC-3’) and ACT-783R (5’-TAC
GAG TCC TTC TGG CCC AT-3’) (Carbone and Kohn, 1999). PCR reaction
conditions were as follows: an initial denaturation step at 96°C for 2 minutes,
followed by 10 cycles of denaturation at 94°C for 30 seconds, primer annealing
at 61°C for 45 seconds and extension at 72°C for 45 seconds. This was
followed by 25 cycles of denaturation at 94°C for 30 seconds, primer annealing
at 61°C for 45 seconds and elongation at 72°C for 45 seconds with an increase
of five seconds per cycle for elongation. The reaction was completed with a
final elongation step at 72°C for 7 minutes.
All PCR products were visualised in 1.5% agarose gels (wt/v) stained
with ethidium bromide, under ultra-violet light. Sizes of PCR amplicons were
estimated by comparison against a 100 bp molecular weight marker (O’
RangeRulerTM 100bp DNA ladder) (Fermentas Life Sciences, Hanover,
U.S.A.). For further DNA sequencing, PCR products were purified through
centri-sep spin columns (Princeton separations, Adelphia, U.S.A.) containing
sephadex G-50 (Sigma Aldrich, St. Louis, U.S.A.) as outlined by the
manufacturer.
DNA sequencing and phylogenetic analysis
The purified PCR products were used as template DNA for cycle
sequencing reactions using the ABI Prism Big Dye Terminator Cycle
sequencing reaction kit v. 3.1 (Applied Biosystems, Foster City, U.S.A.)
following the manufacturers instructions. The same primers as used for the
PCR reactions were also used for sequencing reactions. However, additional
internal primers were used for both the ITS and LSU regions. These were ITS2 (5’-GCT GCG TTC TTC ATC GAT GC-3’) and ITS-3 (5’-GCA TCG ATG
AAG AAC GCA GC-3’) (White et al., 1990) in the case of the ITS and LR3R
(5’-GTC TTG AAA CAC GGA CC-3’) and LR-16 (5’-TTC CAC CCA AAC
ACT CG-3’) in the case of LSU. The precipitated sequencing reactions were
then run on an ABI PRISMTM 3100 Automated DNA sequencer (Applied
Biosystems, Foster City, U.S.A.).
All resulting sequences were analysed with Sequence Navigator v. 1.0.1
(Applied Biosystems, Foster City, U.S.A.). Sequence alignments were done
76
Fungal Diversity
using MAFFT v. 5.667 (Katoh et al., 2005) incorporating the E-INS-i
alignment strategy. A partition homogeneity test (Farris et al., 1994), on all
possible combinations, of 1000 replicates on all informative characters was
conducted in PAUP v. 4.10b (Swofford, 2002) to determine if the DNA
datasets from the four gene regions were combinable.
For phylogenetic analyses, both parsimony and distance analyses were
conducted. For parsimony analysis, most parsimonious trees were generated by
heuristic searches with starting trees obtained through stepwise addition with
the MULPAR function enabled. Tree bisection reconnection (TBR) was
employed as the branch-swapping algorithm. All gaps were coded as missing
data and characters were assigned equal weight. Statistic support for nodes was
obtained by performing 1000 bootstrap replicates.
Modeltest v. 3.04 (Posada and Crandal, 1998) was used to determine the
most appropriate nucleotide substitution model to be applied to the combined
DNA sequence alignment and GTR + I + G was chosen from the Akaike
Information Criterion (AIC) (base frequencies: πA = 0.2387, πC = 0.2588, πG =
0.2780, πT = 0.2245; substitution rates: A/C = 1.4579, A/G = 1.8851, A/T =
1.7352, C/G = 1.2094, C/T = 4.2937, G/T = 1.0000; proportion of invariable
sites (I) = 0.5562; gamma shape distribution parameter = 0.5825). Following
this, a neighbour-joining (NJ) analysis using the GTR + I + G substitution
model was conducted in PAUP. Here, identical sites were removed
proportionally to base frequencies estimated from all sites, rates of invariable
sites assumed to follow a gamma distribution and ties were broken if
encountered. Mycosphaerella lateralis Crous & M.J. Wingf. (anamorph:
Dissoconium dekkeri de Hoog & Hijwegen) was used as an outgroup to root all
trees.
Morphological Studies
Conidia and conidiophores of the undescribed Pseudocercospora sp.
were mounted in lactic acid or bromophenol blue on microscope slides. Leaf
tissue was mounted in Jung Tissue Freezing Medium (Leica Microsystems AG,
Wetzlar, Germany) and sections (10 µm) were cut using a Leica CM 100
Freezing microtome (Leica Microsystems AG, Wetzlar, Germany). Both cross
sections and structures taken from the surface of lesions were examined under
a Zeiss Axioskop light microscope (Carl Zeiss, Jena, Germany) using
differential interference contrast. Fifty measurements of all taxonomically
relevant structures were made at × 1000 magnification. Morphological
characteristics of the unidentified Pseudocercospora sp. from Thailand were
compared with other Pseudocercospora spp. known to occur on Eucalyptus
77
(Crous, 1998; Braun and Dick, 2002). Representative herbarium specimens of
the Thailand Pseudocercospora sp. have been deposited in the National
Collection of Fungi (PREM), Pretoria, South Africa.
Growth characteristics of the undescribed Pseudocercospora sp. on agar
medium were also defined. Plugs (5 mm diam.) of agar were cut from the
actively growing margins of pure cultures of ex-type isolates and transferred to
the centres of 90mm 2% MEA plates. Agar plates were incubated in the dark at
temperatures between 0°C and 35°C at five degree intervals. Colony diameters
were determined every 7 days for one month. Three plates were used per
isolate at each temperature, and the experiment was repeated once. Colony
colours and morphologies were described after one month using the colour
charts of Rayner (1970).
Results
PCR Amplification and analysis of sequence data
Amplification of the LSU, ITS, EF-1α and ACT gene regions for all the
isolates used in this study resulted in amplification products of approximately
600bp. for ITS, 300 bp. for EF-1α, 250 bp. for ACT and 1500 bp. for LSU.
Results of the partition homogeneity test using 1000 replicates resulted in
a P-value > 0.001 for all possible combinations of the LSU, ITS, EF-1α and
ACT DNA sequence alignments. Therefore, DNA sequence alignments of the
ITS, EF-1α, ACT and LSU gene regions were combined. For parsimony
analysis, heuristic searches of the combined DNA sequence alignment resulted
in the retention of six most parsimonious trees, one of which is shown here
(Fig. 4). The tree had a length of 1417 steps (CI = 0.709, RI = 0.8961, HI =
0.291). A total of 2665 characters were evaluated of which 1998 were constant,
153 were parsimony uninformative and 514 were parsimony informative.
Bootstrap searches of the combined data set produced a tree with the same
topology of the most parsimonious tree. Isolates of Mycosphaerella spp. and
isolates of the unknown Pseudocercospora sp. could be resolved into two wellsupported monophyletic clades (Clades I−II) with bootstrap values of 83% and
96%, respectively (Fig. 4).
Clade I could be further resolved into four well-supported sub-clades
(sub-clades 1−4) (Fig. 4). Clade 1 included isolates of P. flavomarginata, P.
paraguayensis (Kobayashi) Crous and P. basiramifera Crous (Bootstrap =
100%), Clade 2 contained isolates of Mycosphaerella fori G.C. Hunter, Crous
& M.J. Wingf., Mycosphaerella gracilis Crous & Alfenas, Pseudocercospora
eucalyptorum Crous, M.J. Wingf., Marasas & B. Sutton, Pseudocercospora
78
Fungal Diversity
pseudoeucalyptorum Crous (Bootstrap = 98%). Clade 3 contained isolates of
Pseudocercospora basitruncata Crous and Pseudocercospora natalensis Crous
& T. Coutinho (Bootstrap = 100%) and Clade 4 contained isolates of
Mycosphaerella colombiensis Crous & M.J. Wingf., Mycosphaerella
crystallina Crous & M.J. Wingf., Mycosphaerella irregulariramosa Crous &
M.J. Wingf., Mycosphaerella heimii Crous and Mycosphaerella heimioides
Crous & M.J. Wingf. (Bootstrap = 100%).
For distance analysis, neighbour-joining analyses yielded a phylogenetic
tree with the same topology as the most parsimonious trees generated by
parsimony analyses (Fig. 5). Here isolates of Mycosphaerella and
Pseudocercospora were resolved into two well-supported monophyletic clades
containing the same isolates as those in the parsimony analysis (Fig. 4).
Furthermore, for the distance analysis, isolates of P. flavomarginata grouped
into a well-supported clade of their own, sister to P. paraguayensis and P.
basiramifera.
Morphology
One of the most obvious distinguishing features of the Pseudocercospora
sp. on E. camaldulensis found in Thailand and Vietnam is the symptoms
associated with the taxon (Fig. 1). These are very distinct angular necrotic
lesions on the leaves, typically surrounded by halos of chlorotic tissue. Lesions
typically bear dense clusters of dark brown conidiophores on the abaxial
surface but these are occasionally also found on the adaxial surfaces of lesions.
Lesions can be very common, mostly on leaves on the lower branches, and
susceptible Eucalyptus clones can be seriously defoliated.
Cultures of the Pseudocercospora sp. grew relatively rapidly on 2%
MEA. Results from growth comparisons at different temperatures showed that
the optimal temperature for growth was 25°C. At 25°C, cultures had a growth
rate of 29 mm in one month. Minimum and maximum temperatures were
between 5−10°C and 30−35°C, respectively. Cultures were pale olivaceousgrey on the surface, and greenish-black in reverse. Cultures exhibited irregular
margins and produced profuse aerial mycelium while colony borders were
generally darker (greenish-black) than the colony centre that tended to become
paler as the culture aged.
Taxonomy
DNA sequence comparison of the Pseudocercospora sp. considered in
the study has shown that the species is different from all other Mycosphaerella
spp. considered during this study. The symptoms associated with this fungus
79
are also very obvious and unique. In addition, the morphology of the fungus is
unlike that of any other Pseudocercospora sp. known on Eucalyptus. We
therefore describe it as a new species as follows:
Pseudocercospora flavomarginata G.C. Hunter, Crous & M.J. Wingf., sp.
nov.
(Figs. 2, 3)
MycoBank number: MB500513.
Etymology: Named for the characteristic chlorotic borders surrounding the angular
necrotic lesions on Eucalyptus camaldulensis leaves.
Conidiophorae dense fasciculatae, brunneae, apicem versus pallidiores, non ramosae,
parietibus crassis, laeves, 0-4-septatae, subcylindricae, rectae ver curvatae, e cellulis stromatis
bene evoluti exorientes, (18−)32−36(−53) × (2−)3−4(−5) µm. Conidia solitaria, recta vel
subfalcata, pallide brunnea, laeves, parietibus crassis, guttulata, acicularia vel obclavata, apice
obtusa, basi rotundata vel longe obconico-truncata, 2-7-septata, (28−)46−54(−90) × (2−)3(−4)
µm. Teleomorpha ignota.
Holotype: Thailand: Chang Gao Province near Pratchinburi: on leaves of Eucalyptus
camaldulensis 2004, M.J. Wingfield (PREM 58952− holotypus; cultura viva ex-types CBS
118841, 118823, 118824).
Additional material examined (paratypes): Thailand, Chang Gao Province near
Pratchinburi on leaves of Eucalyptus camaldulensis, 2004, M.J. Wingfield (CMW 17703,
PREM 58953); Thailand, Chang Gao Province near Pratchinburi on leaves of Eucalyptus
camaldulensis, 2004 M.J. Wingfield (CMW 17707, PREM 58954); Thailand, Chang Gao
Province near Pratchinburi on leaves of Eucalyptus camaldulensis, 2004 M.J. Wingfield
(CMW17708, PREM 58955).
Leaf spots distinct, scattered over leaves, amphigenous, circular to
angular, 3−20 mm diam (Fig. 1), pale to dark brown becoming darker with age,
definite chlorotic margin on abaxial and adaxial leaf surfaces but more obvious
on adaxial leaf surfaces. Mycelium internal and external, pale brown, septate,
branched, thick-walled, smooth to finely verruculose, 2−4 µm diam. Caespituli
amphigenous, predominantly epiphyllous, evenly distributed over lesion,
brown to black on leaves, 74 µm wide and 90 µm high (Figs. 2, 3).
Conidiophores fasciculate, grouped in dense fascicles, conidiophores brown
becoming paler towards apex, unbranched, thick-walled, smooth, 0−4-septate,
subcylindrical, straight to curved, arising from cells of a well developed stroma
(18−)32−36(−53) × (2−)3−4(−5) µm. Stromata well-developed, prominent,
immersed becoming erumpent, brown, 56 µm wide and 47 µm high.
Conidiogenous cells terminal, smooth, thick-walled, pale brown, unbranched,
tapering to a rounded apex, proliferating sympodially or 1−2 times
percurrently, (6−)14−17(−25) × (2−)3(−5) µm. Conidial scars, unthickened
and not darkened. Conidia solitary, straight or slightly curved, pale brown,
smooth, thick-walled, guttulate, acicular to obclavate, apex obtuse, base
rounded to long obconic-truncate, 2−7-septate, (28−)46−54(−90) × (2−)3(−4)
µm. Hilum unthickened, not darkened (Figs. 2, 3). Spermagonium present in
80
Fungal Diversity
Fig. 1. Symptoms of Pseudocercospora flavomarginata on leaves of Eucalyptus
camaldulensis. P. flavomarginata produces necrotic lesions surrounded by characteristic
chlorotic tissue. Lesions bear clusters of dark brown conidiophores on the abaxial and adaxial
lesion surfaces.
81
A
E
B
C
D
F
Fig. 2. Morphological characteristics of Pseudocercospora flavomarginata (PREM 58952,
MycoBank 500513). A−D. Straight or slightly curved, guttulate, acicular to obclavate conidia
with obtuse apex and rounded to long obconic-truncate base. E−F. Fasciculate conidiophores
that are unbranched, thick walled, subcylindrical, straight to curved. Bar = 10 µm.
82
Fungal Diversity
A
D
B
C
Fig. 3. Line drawings of Pseudocercospora flavomarginata. A. Conidia. B. Well-developed,
immersed young spermagonium with developed conidiophores that become lighter towards the
apex. C. Septate, branched, thick walled, smooth, external mycelium. D. Fascicle of
conidiophores. Bar = 10 µm.
83
lesions, well developed, prominent, immersed becoming erumpent 64 µm wide
and 57 µm high. Spermatia not observed. Teleomorph unknown.
Cultures: Cultures 29 mm. diam. on MEA after 1 month at 25°C in the
dark. Colonies pale olivaceous-grey 21’’’’’b (surface) and greenish-black
33’’’’’i (reverse) (Rayner, 1970). Margins regular to irregular, aerial mycelium
profuse. Border darker (greenish-black) than colony centres, which become
paler with age. Colony not sectored and folding absent.
Cardinal temperatures: Minimum 5−10°C, optimum 25°C, maximum
30−35°C.
Hosts: Eucalyptus camaldulensis, E. camaldulensis hybrids.
Distribution: Thailand, Vietnam.
Notes: Pseudocercospora flavomarginata is morphologically and
phylogenetically similar to Pseudocercospora paraguayensis. However, P.
flavomarginata can be distinguished from P. paraguayensis by the very
distinct chlorotic borders around the typically irregular lesions. Conidiophores
are longer and have more septa than those of P. paraguayensis. Furthermore,
conidium development is sympodial and percurrent in P. flavomarginata, but
exclusively sympodial in P. paraguayensis.
Discussion
Pseudocercospora flavomarginata is a fungal pathogen of E.
camaldulensis that is very well-known in Thailand and Vietnam where it
causes leaf spots typically on the lower leaves of young E. camaldulensis trees
(Old et al., 2003). It is thus unfortunate that it has not previously been formally
named. Perhaps this is to some extent due to the fact that there are various
Pseudocercospora spp. on Eucalyptus leaves and DNA sequence data have not
previously been available to reinforce the fact that it represents a novel taxon.
Although Pseudocercospora basiramifera is known to occur in Thailand
on E. camaldulensis and E. pellita F. Muell. (Crous, 1998), it was not
encountered during the present study. Pseudocercospora flavomarginata can
be distinguished from P. basiramifera by the presence of red lesion margins in
P. basiramifera as opposed to the very characteristic chlorotic margins found
around lesions of P. flavomarginata. Furthermore, P. flavomarginata has
smaller conidia that are 2−7-septate and (28−)46−54(−90) × (2−)3(−4) µm in
contrast to conidia of P. basiramifera, which are 3−10-septate and
(35−)50−70(−80) × 2(−3) µm (Crous, 1998).
Various Pseudocercospora spp. other than P. flavomarginata are known
to occur in South-East Asia on Eucalyptus. These include Pseudocercospora
eucalyptorum, P. gracilis Crous & Alfenas, and P. heimioides Crous & M.J.
Wingf. (Crous et al., 1989; Crous and Alfenas, 1995; Crous and Wingfield,
84
Fungal Diversity
1997). Pseudocercospora flavomarginata is common in Thailand and Vietnam
and can be distinguished from P. eucalyptorum, P. gracilis and P. heimioides
by characteristic leaf lesions with prominent yellow borders. This in contrast to
pale to grey-brown lesions with purple borders produced by P. eucalyptorum,
pale brown lesions with red to brown borders formed by P. gracilis, and brown
to absent leaf spots of P. heimioides (Crous et al., 1989; Crous and Alfenas,
1995; Crous and Wingfield, 1997; Crous, 1998).
Phylogenetically, P. flavomarginata is closely related to P.
paraguayensis, which is known from eucalypts in South America (Crous,
1998). Pseudocercospora flavomarginata can be distinguished from P.
paraguayensis by the prominent chlorotic borders around lesions,
conidiophores that are more numerously septate and longer than those of P.
paraguayensis, conidia that are more numerously septate than those of P.
paraguayensis and conidium development that is sympodial and percurrent, in
contrast to the exclusively sympodal development in P. paraguayensis.
DNA sequence comparisons in this study resolved Pseudocercospora
spp. into two well-supported monophyletic clades, supporting results of Crous
et al. (2000), and the recent finding of Ayala-Escobar et al. (2006) that
Pseudocercospora is paraphyletic within Mycosphaerella. While these groups
are very clearly delineated, it is not possible to link them to any distinct
morphological characteristics. However, larger sequence data sets that consider
additional isolates of Cercospora spp. and specifically Pseudocercospora spp.
might in future resolve specific lineages that could be linked to
phylogenetically informative morphological characteristics.
Pseudocercospora crystallina, P. irregulariramosa, P. heimii and P.
heimioides are morphologically similar species that have been shown, based on
ITS sequence data, to group in a single clade (Crous et al., 2000, 2001). These
fungi are regarded as a species complex known as the Mycosphaerella heimii
complex (Crous et al., 2000). Based on DNA sequence results from the four
gene regions in this study, species within the M. heimii complex clustered
together, supporting previous findings. Based on the distance of the M. heimii
complex from its most recent ancestor, it is evident that the divergence from
this node is relatively recent. This suggests that speciation amongst members
of the M. heimii complex has occurred recently.
Eucalyptus spp. are native to Australia, Papua New Guinea, Indonesia
and the Phillipines (Turnbull, 2000). It is possible that P. flavomarginata may
be present in Australia and other areas where Eucalyptus spp. are native.
Further surveys of both natural and commercially propagated Eucalyptus spp.
could result in collections of P. flavomarginata an increase the known host
range of this species.
85
Pseudocercospora flavomarginata CBS118841
100
Pseudocercospora flavomarginata CBS118823
Pseudocercospora flavomarginata CBS118824
100
1
Pseudocercospora paraguayensis CBS111286
98
Pseudocercospora basiramifera CMW5148
72
100
Mycosphaerella fori CMW9095
Mycosphaerella fori CMW9096
Mycosphaerella gracilis CBS243.94
98
74
100
90
Pseudocercospora eucalyptorum CMW5228
2
Pseudocercospora pseudoeucalyptorum CBS114242
Pseudocercospora pseudoeucalyptorum CBS114243
I
Pseudocercospora robusta CMW5151
99
100
83
Pseudocercospora basitruncata CBS114664
Pseudocercospora basitruncata CBS111280
3
Pseudocercospora natalensis CBS111070
100
Mycosphaerella colombiensis CBS110969
Mycosphaerella colombiensis CBS110967
Mycosphaerella crystallina CMW 3042
100
77
Mycosphaerella crystallina CBS681.95
4
Mycosphaerella irregulariramosa CMW 4943
99
Mycosphaerella irregulariramosa CMW 5223
Mycosphaerella heimii CMW4942
Mycosphaerella heimioides CBS111364
100
Mycosphaerella heimioides CMW3046
Mycosphaerella madeirae CBS112895
96
100
Pseudocercospora epispermogonia CBS110750
II
Pseudocercospora epispermogonia CBS110693
Mycosphaerella lateralis CBS110748
10 changes
Fig. 4. Phylogram of Mycosphaerella and Pseudocercospora spp. occurring on Eucalyptus
indicating the phylogenetic placement of Pseudocercospora flavomarginata. One of six most
parsimonious trees generated from a heuristic search of combined DNA alignments of LSU,
ITS, ACT and EF-1α data in PAUP v. 4.0b10 (Length= 1417, CI = 0.79, RI = 0.8961, HI =
0.291). Bootstrap values of 1000 replicates are indicated above branches.
86
Fungal Diversity
NJ
Pseudocercospora flavomarginata CBS118841
100
Pseudocercospora flavomarginata CBS118823
Pseudocercospora flavomarginata CBS118824
100
Pseudocercospora paraguayensis CBS111286
99
98
Pseudocercospora basiramifera CMW5148
100
Mycosphaerella fori CMW9095
Mycosphaerella fori CMW9096
Mycosphaerella gracilis CBS243.94
Pseudocercospora robusta CMW5151
100 100
Pseudocercospora eucalyptorum CMW5228
100
Pseudocercospora pseudoeucalyptorum CBS114242
Pseudocercospora pseudoeucalyptorum CBS114243
99
Pseudocercospora basitruncata CBS114664
Pseudocercospora basitruncata CBS111280
98
100
Pseudocercospora natalensis CBS111070
100
Mycosphaerella colombiensis CBS110969
Mycosphaerella colombiensis CBS110967
Mycosphaerella crystallina CMW3042
100
Mycosphaerella crystallina CBS681.95
Mycosphaerella irregulariramosa CMW4943
Mycosphaerella irregulariramosa CMW5223
Mycosphaerella heimii CMW4942
94
100
Mycosphaerella heimioides CBS111364
Mycosphaerella heimioides CMW3046
Mycosphaerella madeirae CBS112895
99
100
Pseudocercospora epispermogonia CBS110750
Pseudocercospora epispermogonia CBS110693
Mycosphaerella lateralis CBS110748
0.01 substitutions/site
Fig. 5. Neighbour-joining (NJ) tree of Mycosphaerella and Pseudocercospora spp. obtained
from distance analysis of combined LSU, ITS, ACT and EF-1α DNA sequence alignments
using the GTR + I + G DNA substitution model. Bootstrap values after 1000 replicates are
indicated above branches. The tree was rooted to Mycosphaerella lateralis.
87
Eucalyptus camaldulensis is one of the species that is most commonly
grown in South-East Asia. Considering that many commercially propagated
eucalypts are clones or hybrids (Old et al., 2003), it will be important to
consider the host distribution of P. flavomarginata in this area. It is also wellknown that some clones can be particularly susceptible to pathogens while
others, even those relatively closely related, can be resistant. Therefore future
surveys and pathogenicity studies would aid in selecting and propagating
genotypes of Eucalyptus that are tolerant to pathogens such as P.
flavomarginata.
Acknowledgements
We thank the members of the Tree Protection Co-operative Programme (TPCP), the
National Research Foundation (NRF), the Mellon Foundation and the THRIP initiative of the
Department of Trade and Industry (DTI), South Africa for financial Support. Dr. Ken Old and
Dr. Thu assisted in the collection of P. flavomarginata in Vietnam and various students and
colleagues in Thailand are also thanked for assisting us in assembling cultures of this fungus.
References
Aptroot, A. (2006). Mycosphaerella and its Anamorphs: 2. Conspectus of Mycosphaerella.
CBS Biodiversity Series 5, Utrecht, The Netherlands.
Ayala-Escobar, V., Yáñez-Morales, M., de Jesús, Braun, U., Groenewald, J.Z. and Crous, P.W.
(2006). Pseudocercospora opunitae sp. nov., the causal organism of cactus leaf spot in
Mexico. Fungal Diversity 21: 1-9.
Braun, U. and Dick, M.A. (2002). Leaf spot diseases of Eucalypts in New Zealand caused by
Pseudocercospora species. New Zealand Journal of Forestry Science 32: 221−234.
Carbone, I. and Kohn, L.M. (1999). A method for designing primer sets for speciation studies
in filamentous ascomycetes. Mycologia 91: 553-556.
Carnegie, A.J. and Keane, P.J. (1994). Further Mycosphaerella species associated with leaf
diseases of Eucalyptus. Mycological Research 98: 413-418.
Corlett, M. (1991). An annotated list of the published names in Mycosphaerella and
Sphaerella. Mycologia Memoirs 18: 1-328.
Corlett, M. (1995). An annotated list of the published names in Mycosphaerella and
Sphaerella: corrections and additions. Mycotaxon 53: 37-56.
Crous, P.W. (1998). Mycosphaerella spp. and their anamorphs associated with leaf spot
diseases of Eucalyptus. Mycologia Memoir 21: 1-170.
Crous, P.W. and Alfenas, A.C. (1995). Mycosphaerella gracilis and other species of
Mycosphaerella associated with leaf spots of Eucalyptus in Indonesia. Mycologia 87:
121-126.
Crous, P.W., Alfenas, A.C. and Barreto, R.W. (1997). Cercosporoid fungi from Brazil. 1.
Mycotaxon 64: 405-430.
Crous, P.W., Aptroot, A., Kang, J.C., Braun, U. and Wingfield, M.J. (2000). The genus
Mycosphaerella and its anamorphs. Studies in Mycology 45: 107-121.
Crous, P.W. and Braun, U. (1996). Cercosporoid fungi from South Africa. Mycotaxon 57:
233-321.
88
Fungal Diversity
Crous, P.W. and Braun, U. (2001). A reassessment of the Cercospora species described by C.
Chupp: Specimens deposited at BPI, Maryland, USA. Mycotaxon 78: 327-343.
Crous, P.W. and Braun, U. (2003). Mycosphaerella and its anamorphs. 1. Names published in
Cercospora and Passalora. CBS Biodiversity Series 1: 1-571.
Crous, P.W., Groenewald, J.Z., Mansilla, J.P., Hunter, G.C. and Wingfield, M.J. (2004).
Phylogenetic reassessment of Mycosphaerella spp. and their anamorphs occurring on
Eucalyptus. Studies in Mycology 50: 195-214.
Crous, P.W., Hong, L., Wingfield, B.D. and Wingfield, M.J. (2001). ITS rDNA phylogeny of
selected Mycosphaerella species and their anamorphs occurring on Myrtaceae.
Mycological Research 105: 425-431.
Crous, P.W. and Wingfield, M.J. (1997). New species of Mycosphaerella occurring on
Eucalyptus leaves in Indonesia and Africa. Canadian Journal of Botany 75: 781-790.
Crous, P.W., Wingfield, M.J., Mansilla, J.P., Alfenas, A.C. and Groenewald, J.Z. (2006).
Phylogenetic reassessment of Mycosphaerella spp. and their anamorphs occurring on
Eucalyptus. II. Studies in Mycology 55: 99-131.
Crous, P.W., Wingfield, M.J., Marasas, W.F.O. and Sutton, B.C. (1989). Pseudocercospora
eucalyptorum sp. nov. on Eucalyptus leaves. Mycological Research 93: 394-398.
Farris, J.S., Kallersjo, M., Kluge, A.G. and Bult, C. (1994). Testing significance of
Incongruence. Cladistics 10: 315-320.
Hunter, G.C., Roux, J., Wingfield, B.D., Crous, P.W. and Wingfield, M.J. (2004).
Mycosphaerella species causing leaf disease in South African Eucalyptus plantations.
Mycological Research 108: 672-681.
Hunter, G.C., Wingfield, B.D., Crous, P.W. and Wingfield, M.J. (2006). A multi-gene
phylogeny for species of Mycosphaerella occurring on Eucalyptus leaves. Studies in
Mycology 55: 147-161.
Katoh, K., Kuma, K., Toh, H. and Miyata, T. (2005). MAFFT version 5: Improvement in
accuracy of multiple sequence alignment. Nucleic Acid Research 33: 511-518.
Kirschner, R.K., Piepenbring, M. and Chen, C.J. (2004). Some cercosporoid hyphomycetes
from Taiwan, including a new species of Stenella and new reports of Distocercospora
pachyderma and Phacellium papali. Fungal Diversity 17: 57-68.
Maxwell, A., Dell, B., Neumeister-Kemp, H.G. and Hardy, G.E.St.J. (2003). Mycosphaerella
species associated with Eucalyptus in south-western Australia: new species, new
records and a key. Mycological Research 107: 351-359.
Moncalvo, J.M., Wamg, H.H. and Hseu, R.S. (1995). Phylogenetic relationships in Ganoderma
inferred from the internal transcribed spacers and 25S ribosomal DNA sequences.
Mycologia 87: 223-238.
Old, K.M., Wingfield, M.J. and Yuan, Z.Q. (2003). A manual of diseases of Eucalypts in
South-East Asia. Centre for International Research, Indonesia.
Park, R.F. and Keane, P.J. (1982). Leaf diseases of Eucalyptus associated with
Mycosphaerella species. Transactions of the British Mycological Society 79: 101-115.
Posada, D. and Crandall, K.A. (1998). Modeltest: testing the model of DNA substitution.
Bioinformatics 14: 817-818.
Rayner, A.W. (1970). A Mycological colour chart. Commonwealth Mycological Institute,
England.
Schubert, K. and Braun, U. (2005). Taxonomic revision of the genus Cladosporium s.l. 4.
Species reallocated to Asperisporium, Dischloridium, Fusicladium, Passalora,
Pseudoasperisporium and Stenella. Fungal Diversity 20: 187-208.
Swofford, D.L. (2002). PAUP*. Phylogenetic analysis using parsimony (* and other methods).
Version 4.0b10. Sinauer Associates, Sunderland, MA.
89
Turnbull, J.W. (2000). Economic and social importance of eucalypts. In: Diseases and
Pathogens of Eucalypts. (eds. P.J. Keane, G.A. Kile, F.D. Podger and B.N. Brown)
CSIRO Publishing, Australia: 1-9.
Vilgalys, R. and Hester, M. (1990). Rapid genetic identification and mapping of enzymatically
amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology
172: 4238-4246.
White, T.J., Bruns, T., Lee, S. and Taylor, J.W. (1990) Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. In: A Guide to Molecular Methods and
Applications (eds. M.A. Innis, D.H. Gelfand, J.J. Snisky and T.J. White) Academic
Press, U.S.A: 315-322.
(Received 22 February 2006; accepted 16 April 2006)
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